Ribosome‐associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial‐type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U‐tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U‐tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U‐tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U‐tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome‐associated PPRs may selectively activate mRNAs for translation.     

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.     

Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state

Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T→R transition observed in the crystals. Taking the crystal R structure as a reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11° in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9° around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis. Proteins 27:110–117 © 1997 Wiley-Liss, Inc.     

Apoptosis and necrosis: detection, discrimination and phagocytosis.

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.     

High Functional Diversity in Mycobacterium tuberculosis Driven by Genetic Drift and Human Demography

Mycobacterium tuberculosis infects one third of the human world population and kills someone every 15 seconds. For more than a century, scientists and clinicians have been distinguishing between the human- and animal-adapted members of the M. tuberculosis complex (MTBC). However, all human-adapted strains of MTBC have traditionally been considered to be essentially identical. We surveyed sequence diversity within a global collection of strains belonging to MTBC using seven megabase pairs of DNA sequence data. We show that the members of MTBC affecting humans are more genetically diverse than generally assumed, and that this diversity can be linked to human demographic and migratory events. We further demonstrate that these organisms are under extremely reduced purifying selection and that, as a result of increased genetic drift, much of this genetic diversity is likely to have functional consequences. Our findings suggest that the current increases in human population, urbanization, and global travel, combined with the population genetic characteristics of M. tuberculosis described here, could contribute to the emergence and spread of drug-resistant tuberculosis.     

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.     

Structure dissection of human progranulin identifies well-folded granulin/epithelin modules with unique functional activities

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High-resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well-defined three-dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three-dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two beta-hairpins in their N-terminal subdomains, but showed a more flexible C-terminal subdomain. Interestingly, of the well-structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well-characterized human hGrnA offers a unique opportunity for detailed structure-function studies of these important GEM proteins as novel members of mammalian growth factors.     

The structure and binding behavior of the bacterial cell surface layer protein SbsC

Surface layers (S-layers) comprise the outermost cell envelope component of most archaea and many bacteria. Here we present the structure of the bacterial S-layer protein SbsC from Geobacillus stearothermophilus, showing a very elongated and flexible molecule, with strong and specific binding to the secondary cell wall polymer (SCWP). The crystal structure of rSbsC((31-844)) revealed a novel fold, consisting of six separate domains, which are connected by short flexible linkers. The N-terminal domain exhibits positively charged residues regularly spaced along the putative ligand binding site matching the distance of the negative charges on the extended SCWP. Upon SCWP binding, a considerable stabilization of the N-terminal domain occurs. These findings provide insight into the processes of S-layer attachment to the underlying cell wall and self-assembly, and also accommodate the observed mechanical strength, the polarity of the S-layer, and the pronounced requirement for surface flexibility inherent to cell growth and division.     

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angeli's salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.